During infection, pathogenic bacteria selectively activate or repress genes facilitating their survival in the host. The environmental input signals are channeled through regulatory networks utilizing transcriptional and post-transcriptional mechanisms. The effectors of post-transcriptional regulation are primarily non-coding small RNAs (sRNAs). Next generation sequencing can readily identify these sRNAs, however their regulatory targets are extremely difficult to predict. The goal of this proposal is to validate a new tool developed in our laboratory, based on proximity ligation of sRNAs to their targets, catalyzed by T4 RNA ligase. In addition to providing a research tool for the community, our long-term objectives are to define complete regulatory networks controlling P. aeruginosa virulence and establishing links between transcriptional and post- transcriptional components and regulated targets. The project is based on our previous work, identifying a comprehensive set of sRNAs in P. aeruginosa and subsequent assignment a role in virulence for a subset (23) of these regulators. First, we will identify regulatory targets of these sRNAs using the RNA proximity ligation method. In the second phase of the project, we will use a number of molecular tools to identify regulators of sRNA genes, allowing us to connect virulence-associated regulatory networks containing both transcriptional as well as sRNA-based modules. This project represents an early attempt to define function for the vast amount of different sRNAs and assign them a role in bacterial physiology and virulence.